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Effects of Salt Stress on Growth, Antioxidant Enzyme and Phenylalanine Ammonia-Lyase Activities in Jatropha Curcas L

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Effects of Salt Stress on Growth, Antioxidant Enzyme and Phenylalanine Ammonia-Lyase Activities in Jatropha Curcas L Effects of salt stress on growth, antioxidant enzyme and phenylalanine ammonia-lyase activities in Jatropha curcas L. seedlings S. Gao, C. Ouyang, S. Wang, Y. Xu, L. Tang, F. Chen Sichuan Key Laboratory of Resource Biology and Biopharmaceutical Engineering, Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, P.R. China ABSTRACT The effects of increasing NaCl concentrations on biomass, superoxide dismutase (SOD), peroxidase (POD), cata- lase (CAT), and phenylalanine ammonia-lyase (PAL) in Jatropha curcas L. seedlings were investigated. The fresh weights of cotyledons and radicles with increasing NaCl concentrations decreased progressively, and the fresh weight of hypocotyls reached the lowest level at NaCl concentration of 150 mmol and then increased. SOD activity in the cotyledons, hypocotyls and radicles increased gradually up to NaCl concentrations of 150, 200 and 150 mmol, respectively. The highest POD activities in the cotyledons, hypocotyls and radicles were observed at NaCl concen- trations of 150, 200 and 150 mmol, respectively. CAT activity in the cotyledons, hypocotyls and radicles enhanced gradually up to 100, 200 and 150 mmol NaCl concentrations, respectively. Increased PAL activity in the hypocotyls and radicles was linearly and positively correlated with increasing NaCl concentrations, but the peak activity in the cotyledons was observed at NaCl concentration of 150 mmol. Electrophoresis analysis suggested that different pat- terns in SOD and POD isoenzymes depend on NaCl concentrations and organ type, and the staining intensities of these isoforms are consistent with the changes of enzyme activity assayed in solutions. Keywords: ROS-scavenging enzymes; in vitro embryo culture; salt tolerance; isoenzyme pattern Salt stress in soil or water is one of the major antioxidant enzymes such as superoxide dismutase abiotic stresses especially in arid and semi-arid (SOD), peroxidase (POD) and catalase (CAT) have regions and can severely limit plant growth and been considered as a defensive team, whose com- yield. Salt stress can lead to stomatal closure, bined purpose is to protect cells from oxidative which reduces CO2 availability in the leaves and damage (Mittler 2002). Increased SOD, POD and inhibits carbon fixation, exposing chloroplasts to CAT activities are closely related to salt tolerance excessive excitation energy, which in turn could of many plants as reported in various researches increase the generation of reactive oxygen species (Rahnama and Ebrahimzadeh 2005, Azevedo Neto (ROSs) and induce oxidative stress (Parida and et al. 2006, Koca et al. 2007). These findings suggest Das 2005, Parvaiz and Satyawati 2008). that the induction of ROS-scavenging enzymes, ROSs have potential to interact with many cel- such as SOD, POD and CAT, is the most common lular components, causing significant damage to mechanism of salt tolerance for detoxifying ROS membranes and other cellular structures. However, synthesized. an elaborate and highly redundant plant ROS Jatropha curcas L., commonly known as physic network, composed of antioxidant enzymes and nut or purging nut, is a drought resistant shrub or antioxidants, is responsible for maintaining the tree belonging to the family Euphorbiaceae, which levels of ROS under tight control. In plant cell, is cultivated in many tropical and subtropical coun- Supported by the Key Program of the State Science and Technology Commission of China, Project No. 2006BAD07A04, and by the International Cooperation Program, Project No. 2006DFB63400. 374 PLANT SOIL ENVIRON., 54, 2008 (9): 374–381 tries. Different parts of this plant could be used cluding 150 mmol NaCl and 0.5 mmol EDTA. for various purposes, such as energy source, thera- The homogenized suspension was obtained by peutic uses, fertilizer and animal feed. Jatropha centrifuging at 12 000 rpm for 10 min at 4°C. curcas is also well adapted to gravelly, sandy and The supernatant was used as assaying antioxidant saline soils where salinity might be the major enzyme and PAL. Protein content was quantified problem due to limited water supply (Openshaw by the Lowry method and bovine serum albumin 2000). Prior to this study little data known on the as the standard. changes of antioxidant enzymes and isoenzyme SOD activity was determined by the Chen and of these enzymes with respect to salt stress or Pan method (Chen and Pan 1996). One unit of SOD to possible roles in Jatropha curcas seedlings. In was defined as the enzyme activity that inhibited the present study, the effects of increasing NaCl the photoreduction of nitroblue tetrazolium to concentrations on growth, SOD, POD, CAT and blue formazan by 50%. POD activity was performed phenylalanine ammonia-lyase (PAL) in Jatropha according to the Sakharov and Aridilla method curcas L. seedlings were investigated. Moreover, (Sakharov and Aridilla 1999) with slight modifica- patterns of SOD and POD isoenzymes were also tion. One unit of POD activity was expressed as detected, to localize or demonstrate specific SOD the amount of enzyme that produced a change of and POD isoenzymes to specific organs and dif- 1.0 absorbance per min at 470 nm. CAT activity ferent NaCl concentrations. was measured following the change of absorb- ance at 240 nm for 1 min due to H2O2. One unit of CAT activity was expressed as the amount of MATERIAL AND METHODS enzyme needed to reduce 1 µmol of H2O2 per min (Montavon et al. 2007). SOD, POD, and CAT Mature Jatropha curcas L. seeds were harvested activities were expressed as unit per gram fresh in August 2007 from more than 10 individual wild weight (U/g fw). trees in Panzhihua, Sichuan province, China. Seeds Fresh tissues were homogenized with chilled were oven dried, selected and stored in a plastic 50 mmol Tris-HCl (pH 8.8, 1/10, w/v), supple- box (labelled, No. 20070822) and were deposited mented with 0.5 mmol EDTA and 1% polyvinyl at 4°C until use. pyrrolidone. Other steps are the same as above. The seeds were subjected to 70% ethanol for 30 s, PAL activity was measured by monitoring the re- and then to 0.1% mercuric chloride for 8 min. The action product, trans-cinnamate (Hahlbrock and seeds were rinsed several time with sterile distilled Ragg 1975). One unit of PAL activity was defined as water and soaked in sterile water for 24–36 h in the amount causing an increase of 0.01 in A290 per a culture room. Each embryo was dissected from min. PAL activity was expressed as unit per gram the seeds on a clean bench. Three embryos were fresh weight (U/g fw), as well. sown in wide-neck bottles (100 ml) containing SOD gel activity was carried out by the Beauchamp Murashige and Skoog (MS) medium (25 ml) for and Fridovich method with some modifications germination and growth. Embryos were separated (Beauchamp and Fridovich 1971). After comple- into five lots. One lot was allowed to grow on tion of electrophoresis, gels were incubated in MS medium to serve as control. The remaining 50 mmol phosphate buffer (pH 7.5) containing four lots were exposed to four concentrations of 28 µmol riboflavin, 28 mmol N,N,N,N-tetramethyl NaCl: 50, 100, 150 and 200 mmol. MS medium ethylenediamine (TEMED) for 30 min under dark pH was adjusted to 5.8 ± 0.1 prior to autoclaving conditions, followed by washing in distilled water at 121 ± 2°C for 15 min, with 30 g/l sucrose and for 1 min and incubation in the same buffer con- 6 g/l agar powder. Germination experiment was taining 2.45 mmol nitroblue tetrazolium (NBT) carried out at 30°C in a greenhouse. Rotten and for 20 to 30 min exposed to light at room tem- contaminated embryos were removed promptly. perature. Isoenzymes appeared as colorless bands When the cotyledons of seedlings had developed, on a purple background. POD isoenzyme activity cotyledons, hypocotyls and radicles were washed was determined by the procedure described by with distilled water, blotted and immediately frozen Ros Barcelo (1987). Gels, rinsed in water, were in liquid nitrogen or stored at –80°C for analysis. incubated in a solution composed of 0.06% (v/v) Three sets of seedlings were analyzed for each NaCl H2O2, 0.1% (w/v) benzidine and 0.1% (v/v) acetic concentration, with 15 embryos per set. acid at room temperature till brown bands appear, Fresh tissues were homogenized with 50 mmol and the reaction was stopped by rinsing the gels sodium phosphate buffer (pH 7.0, 1/10, w/v) in- with deionized water. PLANT SOIL ENVIRON., 54, 2008 (9): 374–381 375 All treatments were arranged in a completely parameters were consistent with the changes in randomized design with three replicates. All data the fresh weight of cotyledons, hypocotyls and were expressed as means ± SD. Statistical signifi- radicles at all tested NaCl concentrations. Our cance was evaluated with the Student’s t-test, and findings suggested that different changes are related differences were considered significant if P values to the effects of different NaCl concentrations on were 0.05. different organs of seedlings. SOD is one of several important antioxidant enzymes with the ability to repair oxidation dam- RESULTS AND DISCUSSION age caused by ROS. Thus, SOD is considered a key enzyme for maintaining normal physiological Soil salinity is a prevalent abiotic stress for plants. conditions and coping with oxidantive stress in the Growth inhibition is a common response to salin- regulation of intracellular levels of ROS (Mittler ity and plant growth is one of the most important 2002). Effects of NaCl on SOD activity in the coty- agricultural indices of salt stress tolerance as indi- ledons, hypocotyls and radicles were shown in cated by different studies (Parida and Das 2005). Figure 2. SOD activity in the cotyledons increased In order to define salt stress tolerance or sensitiv- significantly with increasing NaCl concentra- ity of seedlings, the effects of NaCl treatment on tions compared to the control, and the maximal fresh weights of the cotyledons, hypocotyls and levels increased by 70.8% at NaCl concentration radicles were tested (Figure 1).
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